Zebra fish (Danio rerio) have been well studied as a model organism in many capacities. Previous proteomic approaches using zebra fish have relied on western blots and 2D gel approaches1 or dimethyl labeling using a morpholino knockdown.2 Another labeling method, using stable isotope labeling by amino acids in cell culture (SILAC), has been used successfully in zebra fish.3 Expanding upon this method, Konzer et al.4 developed a Lys-6-containing diet for complete SILAC labeling in zebra fish for the investigation of proteins involved in embryonic heart development.
After hatching, zebra fish were fed with a heavy diet containing 13C6 lysine (Lys-6) and supplemented with regular nonlabeled fish food for the first 6 weeks. After the fish reached adulthood, they were anesthetized and dissected. For mass spectrometry, organs were homogenized and lysed. A rough estimation of the protein content was estimated. Proteins were then precipitated and reduced prior to SDS-PAGE separation and Colloidal Blue staining.
Proteins were analyzed on an LTQ-Orbitrap XL or a LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific), which was coupled to a 1200 nanoflow or Proxeon LC system (Agilent). The MaxQuant software package (version 22.214.171.124. and 126.96.36.199) was used to analyze raw MS data.
The Andromeda search engine was searched against a zebra fish FASTA database (IPI 3.67, 38,404 entries; Uniprot release 2012_02, 39,559 entries). MaxQuant was also employed for peptides with a maximum of two missed cleavages. GFP expression from the zebra fish line Tg(myl7:EGFP-HsHRAS)s883 helped to demonstrate this stable isotope labeling method for large-scale quantitative analysis by comparing embryonic hearts 72 and 120 h after fertilization. A total of 1,955 proteins were quantified at both time points using a false discovery rate of 1%.
A morpholino-based ALCAM (activated leukocyte cell adhesion molecule) knockdown was also investigated during development. Upregulation of 18 markers during development was accompanied by downregulation of proteins, including the Integrin receptor β, the Ras-like protein Rac1a, and the lipid anchor protein Paralemmin. This work provided a great resource for developmental studies in zebra fish and opened the door for additional quantitative studies using stable isotope labeling.
1. Lucitt, M.B., et al. (2008) ‘Analysis of the zebrafish proteome during embryonic development‘, Molecular and Cellular Proteomics, 7 (5), (pp. 981–994)
2. Lemeer, S., et al. (2007) ‘Protein-tyrosine kinase activity profiling in knock down zebrafish embryos‘, PLoS One, 2 (7), (p. e581)
3. Westman-Brinkmalm, A., et al. (2011) ‘SILAC zebrafish for quantitative analysis of protein turnover and tissue regeneration‘, Journal of Proteomics, 75 (2), (pp. 425–434)
4. Konzer, A., et al. (2013) ‘Stable isotope labelling in zebrafish allows in vivo monitoring of cardiac morphogenesis‘, Molecular and Cellular Proteomics, February 14, 2013, doi: /10.1074/mcp.M111.015594